Crystallographic fragment screening delivers diverse chemical scaffolds for Zika virus NS2B-NS3 protease inhibitor development.

Zika virus (ZIKV) infections cause microcephaly in new-borns and Guillain-Barre syndrome in adults raising a significant global public health concern, yet no vaccines or antiviral drugs have been developed to prevent or treat ZIKV infections. The viral protease NS3 and its co-factor NS2B are essential for the cleavage of the Zika polyprotein precursor into individual structural and non-structural proteins and is therefore an attractive drug target. Generation of a robust crystal system of co-expressed NS2B-NS3 protease has enabled us to perform a crystallographic fragment screening campaign with 1076 fragments. 48 binders with diverse chemical scaffolds were identified in the active site of the protease, with another 6 fragment hits observed in a potential allosteric binding site. Our work provides potential starting points for the development of potent NS2B-NS3 protease inhibitors. Furthermore, we have structurally characterized a potential allosteric binding pocket, identifying opportunities for allosteric inhibitor development.

Studies on the selectivity of the SARS-CoV-2 papain-like protease reveal the importance of the P2' proline of the viral polyprotein.

The SARS-CoV-2 papain-like protease (PLpro) is an antiviral drug target that catalyzes the hydrolysis of the viral polyproteins pp1a/1ab, so releasing the non-structural proteins (nsps) 1-3 that are essential for the coronavirus lifecycle. The LXGG↓X motif in pp1a/1ab is crucial for recognition and cleavage by PLpro. We describe molecular dynamics, docking, and quantum mechanics/molecular mechanics (QM/MM) calculations to investigate how oligopeptide substrates derived from the viral polyprotein bind to PLpro. The results reveal how the substrate sequence affects the efficiency of PLpro-catalyzed hydrolysis. In particular, a proline at the P2' position promotes catalysis, as validated by residue substitutions and mass spectrometry-based analyses. Analysis of PLpro catalyzed hydrolysis of LXGG motif-containing oligopeptides derived from human proteins suggests that factors beyond the LXGG motif and the presence of a proline residue at P2' contribute to catalytic efficiency, possibly reflecting the promiscuity of PLpro. The results will help in identifying PLpro substrates and guiding inhibitor design.

Mass spectrometric assays monitoring the deubiquitinase activity of the SARS-CoV-2 papain-like protease inform on the basis of substrate selectivity and have utility for substrate identification.

The SARS-CoV-2 papain-like protease (PLpro) and main protease (Mpro) are nucleophilic cysteine enzymes that catalyze hydrolysis of the viral polyproteins pp1a/1ab. By contrast with Mpro, PLpro is also a deubiquitinase (DUB) that accepts post-translationally modified human proteins as substrates. Here we report studies on the DUB activity of PLpro using synthetic Nε-lysine-branched oligopeptides as substrates that mimic post-translational protein modifications by ubiquitin (Ub) or Ub-like modifiers (UBLs), such as interferon stimulated gene 15 (ISG15). Mass spectrometry (MS)-based assays confirm the DUB activity of isolated recombinant PLpro. They reveal that the sequence of both the peptide fragment derived from the post-translationally modified protein and that derived from the UBL affects PLpro catalysis; the nature of substrate binding in the S sites appears to be more important for catalytic efficiency than binding in the S' sites. Importantly, the results reflect the reported cellular substrate selectivity of PLpro, i.e. human proteins conjugated to ISG15 are better substrates than those conjugated to Ub or other UBLs. The combined experimental and modelling results imply that PLpro catalysis is affected not only by the identity of the substrate residues binding in the S and S' sites, but also by the substrate fold and the conformational dynamics of the blocking loop 2 of the PLpro:substrate complex. Nε-Lysine-branched oligopeptides thus have potential to help the identification of PLpro substrates. More generally, the results imply that MS-based assays with Nε-lysine-branched oligopeptides have potential to monitor catalysis by human DUBs and hence to inform on their substrate preferences.

Dysfunction of ubiquitin protein ligase MYCBP2 leads to cell resilience in human breast cancers.

Breast cancer is the most common type of cancer among women worldwide, and it is estimated that 294 000 new diagnoses and 37 000 deaths will occur each year in the United States alone by 2030. Large-scale genomic studies have identified a number of genetic loci with alterations in breast cancer. However, identification of the genes that are critical for tumorgenicity still remains a challenge. Here, we perform a comprehensive functional multi-omics analysis of somatic mutations in breast cancer and identify previously unknown key regulators of breast cancer tumorgenicity. We identify dysregulation of MYCBP2, an E3 ubiquitin ligase and an upstream regulator of mTOR signaling, is accompanied with decreased disease-free survival. We validate MYCBP2 as a key target through depletion siRNA using in vitro apoptosis assays in MCF10A, MCF7 and T47D cells. We demonstrate that MYCBP2 loss is associated with resistance to apoptosis from cisplatin-induced DNA damage and cell cycle changes, and that CHEK1 inhibition can modulate MYCBP2 activity and caspase cleavage. Furthermore, we show that MYCBP2 knockdown is associated with transcriptomic responses in TSC2 and in apoptosis genes and interleukins. Therefore, we show that MYCBP2 is an important genetic target that represents a key node regulating multiple molecular pathways in breast cancer corresponding with apparent drug resistance in our study.

Development of a Nickel-Catalyzed N-N Coupling for the Synthesis of Hydrazides.

A nickel-catalyzed N-N cross-coupling for the synthesis of hydrazides is reported. O-Benzoylated hydroxamates were efficiently coupled with a broad range of aryl and aliphatic amines via nickel catalysis to form hydrazides in an up to 81% yield. Experimental evidence implicates the intermediacy of electrophilic Ni-stabilized acyl nitrenoids and the formation of a Ni(I) catalyst via silane-mediated reduction. This report constitutes the first example of an intermolecular N-N coupling compatible with secondary aliphatic amines.

Histone H3 lysine 27 crotonylation mediates gene transcriptional repression in chromatin.

Histone lysine acylation, including acetylation and crotonylation, plays a pivotal role in gene transcription in health and diseases. However, our understanding of histone lysine acylation has been limited to gene transcriptional activation. Here, we report that histone H3 lysine 27 crotonylation (H3K27cr) directs gene transcriptional repression rather than activation. Specifically, H3K27cr in chromatin is selectively recognized by the YEATS domain of GAS41 in complex with SIN3A-HDAC1 co-repressors. Proto-oncogenic transcription factor MYC recruits GAS41/SIN3A-HDAC1 complex to repress genes in chromatin, including cell-cycle inhibitor p21. GAS41 knockout or H3K27cr-binding depletion results in p21 de-repression, cell-cycle arrest, and tumor growth inhibition in mice, explaining a causal relationship between GAS41 and MYC gene amplification and p21 downregulation in colorectal cancer. Our study suggests that H3K27 crotonylation signifies a previously unrecognized, distinct chromatin state for gene transcriptional repression in contrast to H3K27 trimethylation for transcriptional silencing and H3K27 acetylation for transcriptional activation.

In vitro selection of macrocyclic peptide inhibitors containing cyclic γ2,4-amino acids targeting the SARS-CoV-2 main protease.

γ-Amino acids can play important roles in the biological activities of natural products; however, the ribosomal incorporation of γ-amino acids into peptides is challenging. Here we report how a selection campaign employing a non-canonical peptide library containing cyclic γ2,4-amino acids resulted in the discovery of very potent inhibitors of the SARS-CoV-2 main protease (Mpro). Two kinds of cyclic γ2,4-amino acids, cis-3-aminocyclobutane carboxylic acid (γ1) and (1R,3S)-3-aminocyclopentane carboxylic acid (γ2), were ribosomally introduced into a library of thioether-macrocyclic peptides. One resultant potent Mpro inhibitor (half-maximal inhibitory concentration = 50 nM), GM4, comprising 13 residues with γ1 at the fourth position, manifests a 5.2 nM dissociation constant. An Mpro:GM4 complex crystal structure reveals the intact inhibitor spans the substrate binding cleft. The γ1 interacts with the S1' catalytic subsite and contributes to a 12-fold increase in proteolytic stability compared to its alanine-substituted variant. Knowledge of interactions between GM4 and Mpro enabled production of a variant with a 5-fold increase in potency.

AI-Driven Discovery of SARS-CoV-2 Main Protease Fragment-like Inhibitors with Antiviral Activity In Vitro.

SARS-CoV-2 is the causative agent of COVID-19 and is responsible for the current global pandemic. The viral genome contains 5 major open reading frames of which the largest ORF1ab codes for two polyproteins, pp1ab and pp1a, which are subsequently cleaved into 16 nonstructural proteins (nsp) by two viral cysteine proteases encoded within the polyproteins. The main protease (Mpro, nsp5) cleaves the majority of the nsp's, making it essential for viral replication and has been successfully targeted for the development of antivirals. The first oral Mpro inhibitor, nirmatrelvir, was approved for treatment of COVID-19 in late December 2021 in combination with ritonavir as Paxlovid. Increasing the arsenal of antivirals and development of protease inhibitors and other antivirals with a varied mode of action remains a priority to reduce the likelihood for resistance emerging. Here, we report results from an artificial intelligence-driven approach followed by in vitro validation, allowing the identification of five fragment-like Mpro inhibitors with IC50 values ranging from 1.5 to 241 µM. The three most potent molecules (compounds 818, 737, and 183) were tested against SARS-CoV-2 by in vitro replication in Vero E6 and Calu-3 cells. Compound 818 was active in both cell models with an EC50 value comparable to its measured IC50 value. On the other hand, compounds 737 and 183 were only active in Calu-3, a preclinical model of respiratory cells, showing selective indexes twice as high as those for compound 818. We also show that our in silico methodology was successful in identifying both reversible and covalent inhibitors. For instance, compound 818 is a reversible chloromethylamide analogue of 8-methyl-γ-carboline, while compound 737 is an N-pyridyl-isatin that covalently inhibits Mpro. Given the small molecular weights of these fragments, their high binding efficiency in vitro and efficacy in blocking viral replication, these compounds represent good starting points for the development of potent lead molecules targeting the Mpro of SARS-CoV-2.

Structural Analysis of Genomic and Proteomic Signatures Reveal Dynamic Expression of Intrinsically Disordered Regions in Breast Cancer and Tissue.

Structural features of proteins capture underlying information about protein evolution and function, which enhances the analysis of proteomic and transcriptomic data. Here we develop Structural Analysis of Gene and protein Expression Signatures (SAGES), a method that describes expression data using features calculated from sequence-based prediction methods and 3D structural models. We used SAGES, along with machine learning, to characterize tissues from healthy individuals and those with breast cancer. We analyzed gene expression data from 23 breast cancer patients and genetic mutation data from the COSMIC database as well as 17 breast tumor protein expression profiles. We identified prominent expression of intrinsically disordered regions in breast cancer proteins as well as relationships between drug perturbation signatures and breast cancer disease signatures. Our results suggest that SAGES is generally applicable to describe diverse biological phenomena including disease states and drug effects.

Pseudo-temporal dynamics of chemoresistant triple negative breast cancer cells reveal EGFR/HER2 inhibition as synthetic lethal during mid-neoadjuvant chemotherapy.

In the absence of targetable hormonal axes, chemoresistance for triple-negative breast cancer (TNBC) often compromises patient outcomes. To investigate the underlying tumor dynamics, we performed trajectory analysis on the single-nuclei RNA-seq (snRNA-seq) of chemoresistant tumor clones during neoadjuvant chemotherapy (NAC). It revealed a common tumor trajectory across multiple patients with HER2-like expansions during NAC. Genome-wide CRISPR-Cas9 knock-out on mammary epithelial cells revealed chemosensitivity-promoting knock-outs were up-regulated along the tumor trajectory. Furthermore, we derived a consensus gene signature of TNBC chemoresistance by comparing the trajectory transcriptome with chemoresistant transcriptomes from TNBC cell lines and poor prognosis patient samples to predict FDA-approved drugs, including afatinib (pan-HER inhibitor), targeting the consensus signature. We validated the synergistic efficacy of afatinib and paclitaxel in chemoresistant TNBC cells and confirmed pharmacological suppression of the consensus signature. The study provides a dynamic model of chemoresistant tumor transcriptome, and computational framework for pharmacological intervention.

Alkyne Derivatives of SARS-CoV-2 Main Protease Inhibitors Including Nirmatrelvir Inhibit by Reacting Covalently with the Nucleophilic Cysteine.

Nirmatrelvir (PF-07321332) is a nitrile-bearing small-molecule inhibitor that, in combination with ritonavir, is used to treat infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Nirmatrelvir interrupts the viral life cycle by inhibiting the SARS-CoV-2 main protease (Mpro), which is essential for processing viral polyproteins into functional nonstructural proteins. We report studies which reveal that derivatives of nirmatrelvir and other Mpro inhibitors with a nonactivated terminal alkyne group positioned similarly to the electrophilic nitrile of nirmatrelvir can efficiently inhibit isolated Mpro and SARS-CoV-2 replication in cells. Mass spectrometric and crystallographic evidence shows that the alkyne derivatives inhibit Mpro by apparent irreversible covalent reactions with the active site cysteine (Cys145), while the analogous nitriles react reversibly. The results highlight the potential for irreversible covalent inhibition of Mpro and other nucleophilic cysteine proteases by alkynes, which, in contrast to nitriles, can be functionalized at their terminal position to optimize inhibition and selectivity, as well as pharmacodynamic and pharmacokinetic properties.

TET1 and TDG Suppress Inflammatory Response in Intestinal Tumorigenesis: Implications for Colorectal Tumors With the CpG Island Methylator Phenotype.

BACKGROUND & AIMS: Aberrant DNA methylation is frequent in colorectal cancer (CRC), but underlying mechanisms and pathologic consequences are poorly understood. METHODS: We disrupted active DNA demethylation genes Tet1 and/or Tdg from ApcMin mice and characterized the methylome and transcriptome of colonic adenomas. Data were compared to human colonic adenocarcinomas (COAD) in The Cancer Genome Atlas. RESULTS: There were increased numbers of small intestinal adenomas in ApcMin mice expressing the TdgN151A allele, whereas Tet1-deficient and Tet1/TdgN151A-double heterozygous ApcMin colonic adenomas were larger with features of erosion and invasion. We detected reduction in global DNA hypomethylation in colonic adenomas from Tet1- and Tdg-mutant ApcMin mice and hypermethylation of CpG islands in Tet1-mutant ApcMin adenomas. Up-regulation of inflammatory, immune, and interferon response genes was present in Tet1- and Tdg-mutant colonic adenomas compared to control ApcMin adenomas. This up-regulation was also seen in murine colonic organoids and human CRC lines infected with lentiviruses expressing TET1 or TDG short hairpin RNA. A 127-gene inflammatory signature separated colonic adenocarcinomas into 4 groups, closely aligned with their microsatellite or chromosomal instability and characterized by different levels of DNA methylation and DNMT1 expression that anticorrelated with TET1 expression. Tumors with the CpG island methylator phenotype (CIMP) had concerted high DNMT1/low TET1 expression. TET1 or TDG knockdown in CRC lines enhanced killing by natural killer cells. CONCLUSIONS: Our findings reveal a novel epigenetic regulation, linked to the type of genomic instability, by which TET1/TDG-mediated DNA demethylation decreases methylation levels and inflammatory/interferon/immune responses. CIMP in CRC is triggered by an imbalance of methylating activities over demethylating activities. These mice represent a model of CIMP CRC.

Multi-omics-based analysis of high grade serous ovarian cancer subtypes reveals distinct molecular processes linked to patient prognosis.

Despite advancements in treatment, high-grade serous ovarian cancer (HGSOC) is still characterized by poor patient outcomes. To understand the molecular heterogeneity of this disease, which underlies the challenge in selecting optimal treatments for HGSOC patients, we have integrated genomic, transcriptomic, and epigenetic information to identify seven new HGSOC subtypes using a multiscale clustering method. These subtypes not only have significantly distinct overall survival, but also exhibit unique patterns of gene expression, microRNA expression, DNA methylation, and copy number alterations. As determined by our analysis, patients with similar clinical outcomes have distinct profiles of activated or repressed cellular processes, including cell cycle, epithelial-to-mesenchymal transition, immune activation, interferon response, and cilium organization. Furthermore, we performed a multiscale gene co-expression network analysis to identify subtype-specific key regulators and predicted optimal targeted therapies based on subtype-specific gene expression. In summary, this study provides new insights into the cellular heterogeneity of the HGSOC genomic, epigenetic, and transcriptomic landscapes and provides a basis for future studies into precision medicine for HGSOC patients.

Single-Cell RNA Sequencing Reveals New Basic and Translational Insights in the Cystic Fibrosis Lung.

Cystic fibrosis (CF) is a multisystemic, autosomal recessive disorder caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene, with the majority of morbidity and mortality extending from lung disease. Single-cell RNA sequencing (scRNA-seq) has been leveraged in the lung and elsewhere in the body to articulate discrete cell populations, describing cell types, states, and lineages as well as their roles in health and disease. In this translational review, we provide an overview of the current applications of scRNA-seq to the study of the normal and CF lungs, allowing the beginning of a new cellular and molecular narrative of CF lung disease, and we highlight some of the future opportunities to further leverage scRNA-seq and complementary single-cell technologies in the study of CF as we bridge from scientific understanding to clinical application.

Selective Inhibitors of Autophagy Reveal New Link between the Cell Cycle and Autophagy and Lead to Discovery of Novel Synergistic Drug Combinations.

Autophagy is a conserved metabolic pathway that is central to many diseases. Recently, there has been a lot of interest in targeting autophagy with small molecule inhibitors as a possible therapeutic strategy. However, many of the compounds used for autophagy are nonselective. Here, we explored the inhibition of autophagy in pancreatic cancer cells using established selective small molecule inhibitors and discovered an unexpected link between the autophagy pathway and progression through the cell cycle. Our findings revealed that treatments with inhibitors that have different autophagy pathway targets block cell replication and activate other metabolic pathways to compensate for the blockade in autophagy. An unbiased screen looking for known drugs that might synergize with autophagy inhibition revealed new combination treatments that might provide a blueprint for therapeutic approaches to pancreatic cancer. The drugs quizartinib and THZ1 showed a strong synergistic effect in pancreatic cells with autophagy inhibition.

LINE-1 promotes tumorigenicity and exacerbates tumor progression via stimulating metabolism reprogramming in non-small cell lung cancer.

BACKGROUND: Long Interspersed Nuclear Element-1 (LINE-1, L1) is increasingly regarded as a genetic risk for lung cancer. Transcriptionally active LINE-1 forms a L1-gene chimeric transcript (LCTs), through somatic L1 retrotransposition (LRT) or L1 antisense promoter (L1-ASP) activation, to play an oncogenic role in cancer progression. METHODS: Here, we developed Retrotransposon-gene fusion estimation program (ReFuse), to identify and quantify LCTs in RNA sequencing data from TCGA lung cancer cohort (n = 1146) and a single cell RNA sequencing dataset then further validated those LCTs in an independent cohort (n = 134). We next examined the functional roles of a cancer specific LCT (L1-FGGY) in cell proliferation and tumor progression in LUSC cell lines and mice. RESULTS: The LCT events correspond with specific metabolic processes and mitochondrial functions and was associated with genomic instability, hypomethylation, tumor stage and tumor immune microenvironment (TIME). Functional analysis of a tumor specific and frequent LCT involving FGGY (L1-FGGY) reveal that the arachidonic acid (AA) metabolic pathway was activated by the loss of FGGY through the L1-FGGY chimeric transcript to promote tumor growth, which was effectively targeted by a combined use of an anti-HIV drug (NVR) and a metabolic inhibitor (ML355). Lastly, we identified a set of transcriptomic signatures to stratify the LUSC patients with a higher risk for poor outcomes who may benefit from treatments using NVR alone or combined with an anti-metabolism drug. CONCLUSIONS: This study is the first to characterize the role of L1 in metabolic reprogramming of lung cancer and provide rationale for L1-specifc prognosis and potential for a therapeutic strategy for treating lung cancer. TRIAL REGISTRATION: Study on the mechanisms of the mobile element L1-FGGY promoting the proliferation, invasion and immune escape of lung squamous cell carcinoma through the 12-LOX/Wnt pathway, Ek2020111. Registered 27 March 2020 - Retrospectively registered.

Penicillin Derivatives Inhibit the SARS-CoV-2 Main Protease by Reaction with Its Nucleophilic Cysteine.

The SARS-CoV-2 main protease (Mpro) is a medicinal chemistry target for COVID-19 treatment. Given the clinical efficacy of ß-lactams as inhibitors of bacterial nucleophilic enzymes, they are of interest as inhibitors of viral nucleophilic serine and cysteine proteases. We describe the synthesis of penicillin derivatives which are potent Mpro inhibitors and investigate their mechanism of inhibition using mass spectrometric and crystallographic analyses. The results suggest that ß-lactams have considerable potential as Mpro inhibitors via a mechanism involving reaction with the nucleophilic cysteine to form a stable acyl-enzyme complex as shown by crystallographic analysis. The results highlight the potential for inhibition of viral proteases employing nucleophilic catalysis by ß-lactams and related acylating agents.

Mass Spectrometric Assays Reveal Discrepancies in Inhibition Profiles for the SARS-CoV-2 Papain-Like Protease.

The two SARS-CoV-2 proteases, i. e. the main protease (Mpro ) and the papain-like protease (PLpro ), which hydrolyze the viral polypeptide chain giving functional non-structural proteins, are essential for viral replication and are medicinal chemistry targets. We report a high-throughput mass spectrometry (MS)-based assay which directly monitors PLpro catalysis in vitro. The assay was applied to investigate the effect of reported small-molecule PLpro inhibitors and selected Mpro inhibitors on PLpro catalysis. The results reveal that some, but not all, PLpro inhibitor potencies differ substantially from those obtained using fluorescence-based assays. Some substrate-competing Mpro inhibitors, notably PF-07321332 (nirmatrelvir) which is in clinical development, do not inhibit PLpro . Less selective Mpro inhibitors, e. g. auranofin, inhibit PLpro , highlighting the potential for dual PLpro /Mpro inhibition. MS-based PLpro assays, which are orthogonal to widely employed fluorescence-based assays, are of utility in validating inhibitor potencies, especially for inhibitors operating by non-covalent mechanisms.

Female gender increases risk of stroke and readmission after carotid endarterectomy and carotid artery stenting.

OBJECTIVE: Carotid endarterectomy (CEA) has historically demonstrated a higher rate of perioperative adverse events for female patients. However, recent evidence suggests similar outcomes for CEA between genders. In contrast, fewer studies have examined gender in carotid artery stenting (CAS). Using contemporary data from the American College of Surgeons National Surgical Quality Improvement Program database, we aim to determine if gender impacts differences in postoperative complications in patients who undergo CEA or CAS. METHODS: The American College of Surgeons National Surgical Quality Improvement Program database was queried from 2005 to 2017 using Current Procedural Terminology and International Classification of Diseases codes for retrospective review. Patients with carotid intervention (CEA or CAS) were stratified into asymptomatic vs symptomatic cohorts to determine the effect of gender on 30-day postoperative outcomes. Symptomatic patients were defined as those with perioperative transient cerebral ischemic attack or stenosis of carotid artery with cerebral infarction. Descriptive statistics were calculated. Risk-adjusted odds of 30-day postoperative outcomes were calculated using multivariate regression analysis with fixed effects for age, race, and comorbidities. RESULTS: There were 106,568 patients with CEA or CAS (104,412 CEA and 2156 CAS). The average age was 70.9 years, and female patients accounted for 39.9% of the population. For asymptomatic patients that underwent CEA or CAS, female gender was associated with significantly higher rates of cerebrovascular accident (CVA)/stroke (13%; P = .005), readmission (10%; P = .004), bleeding complication (32%; P = .001), and urinary tract infection (54%; P = .001), as well as less infection (26%; P = .001). In the symptomatic cohort, female gender was associated with significantly higher rates of CVA/stroke (32%; P = .034), bleeding complication (203%; P = .001), and urinary tract infection (70%; P = .011), whereas female gender was associated with a lower rate of pneumonia (39%; P = .039). Subset analysis found that, compared with male patients, female patients <75 years old have an increased rate of CVA/stroke (21%; P = .001) and readmission (15%; P < .001), whereas female patients ≥75 years old did not. In asymptomatic and symptomatic patients that underwent CEA, female gender was associated with significantly higher rates of CVA/stroke (13%; P = .006 and 31%; P = .044, respectively), but this finding was not present in patients undergoing CAS. CONCLUSIONS: In patients undergoing carotid intervention, female gender was associated with significantly increased rates of postoperative CVA/stroke in the asymptomatic and symptomatic cohorts as well as readmission in the asymptomatic cohort. Female gender was associated with higher rates of CVA/stroke following CEA, but not CAS. We recommend that randomized control trials ensure adequate representation of female patients to better understand gender-based disparities in carotid intervention.

An automatic pipeline for the design of irreversible derivatives identifies a potent SARS-CoV-2 Mpro inhibitor.

Designing covalent inhibitors is increasingly important, although it remains challenging. Here, we present covalentizer, a computational pipeline for identifying irreversible inhibitors based on structures of targets with non-covalent binders. Through covalent docking of tailored focused libraries, we identify candidates that can bind covalently to a nearby cysteine while preserving the interactions of the original molecule. We found âˆ¼11,000 cysteines proximal to a ligand across 8,386 complexes in the PDB. Of these, the protocol identified 1,553 structures with covalent predictions. In a prospective evaluation, five out of nine predicted covalent kinase inhibitors showed half-maximal inhibitory concentration (IC50) values between 155 nM and 4.5 µM. Application against an existing SARS-CoV Mpro reversible inhibitor led to an acrylamide inhibitor series with low micromolar IC50 values against SARS-CoV-2 Mpro. The docking was validated by 12 co-crystal structures. Together these examples hint at the vast number of covalent inhibitors accessible through our protocol.